Project Summary: In numerous processes including development and metastasis, cells can move in microtracks within the 3D microenvironment. These microtracks are formed by cells themselves through the use of matrix metalloproteinases that degrade matrix, or microtracks can exist as a product of the natural architecture of organs. While microtrack migration occurs in vivo, little is known about the specific mechanisms that cells employ to move in microtracks. We have developed a unique platform using microfabrication to recreate these microtracks in vitro by micromolding collagen. Microtracks can be made in various sizes, and they can be patterned into multiple different shapes including tapered channels and bifurcated channels. Our microfabricated microtracks are structurally indistinguishable from tracks found in vitro and in vivo. Moreover, they offer a distinct advantage over other PDMS-based platforms because the collagen is amenable to cell adhesion on all 4 walls of the track, the fibrous walls of the microtrack can be deformed by cells, and the tracks more closely mimic the mechanical and chemical properties found in vivo. Importantly, our work to-date has shown that the mechanisms driving movement in microtracks are not the same as those mediating cell migration on 2D substrates or in unmolded collagen. Here, we propose to build upon two of our major prior findings, which are that: 1. Vinculin is required for microtrack movement, 2. Cellular confinement alters migration and correlates with cell metabolism. Using this novel microtrack platform in concert with engineered probes to monitor adhesion and cellular energy, optogenetic probes to alter cell contractility and cellular protrusions, and novel force measurement techniques, we will investigate the molecular mechanisms driving cell migration and decision-making during migration in microtracks with a focus on adhesion dynamics and cellular energetics. In Aim 1, we investigate the role of focal adhesion dynamics and tension, focusing on vinculin-talin-actin interactions based on our preliminary showing vinculin mediates unidirectional motion. We will investigate the linkage between vinculin, talin and actin, and we will probe the force transmission occurring at the sites of cell-matrix adhesion. In Aim 2, we will investigate how cellular energetics and the availability of nutrients affects migration and migration decisions in confined spaces. Based on our prior work indicating that the extracellular matrix structure alters ATP utilization, we hypothesize that increased confinement will increase the energetic needs of the cell. In Aim 3, we will investigate the molecular and mechanical mechanisms governing cell migration decisions. Constructs designed to disrupt force transmission between the cell and the matrix and pharmacological interventions will be used to assess the effects of cell contractility and cell stiffness on cellular energy utilization, adhesion, and migration direction decisions in microtracks. Our understanding of metabolism is rapidly developing, and as such, therapeutics targeting metabolic pathways are emerging. Connecting migration behaviors to metabolism offers a potential new point of intervention in disease.